This application is the national phase of PCT Patent Application GB97/02790 filed on Oct. 9, 1997; which claims priority to UK Provisional Application No. GB 9621129.7, filed on Oct. 10, 1996.
1. Technical Field of the Invention
This invention relates to a method of detecting a predisposition to, and determining risk of, sight-threatening diabetic retinopathy. The invention also provides diagnostic kits for the assessment of risk of developing sight-threatening diabetic retinopathy.
2. Description of the Prior Art
Insulin dependent (Type I) and non-insulin dependent (Type II) diabetes mellitus are distinct diseases and patients with either form of the disease are at risk of developing microvascular and macrovascular complications such as neuropathy, nephropathy, retinopathy, atherosclerosis and cardiovascular disease. These complications are a major clinical burden in diabetes, but their pathogenesis is not well understood. Susceptibility to diabetic complications has been reported to be inherited independently of diabetes itself. [Seaquist et al., 1989; Ko et al., 1995].
In the United States there are approximately 16 million diabetics, of which it is estimated that only half are diagnosed [CDC, 1995]. Retinal disease (retinopathy) is one of several complications of diabetes and is primarily the result of disruption of small blood vessels, such as capillary leakage, destruction or occlusion of capillaries leading to ischemia, and the uncontrolled growth of new vessels. In many situations, these changes affect the macula, which is the area of the retina that is specialized to color and visual acuity. All of these are consequences of retinal capillary closure or leakage and are not thought of as retinal inflammation.
Characteristic anatomical and physiological changes occur in the retinal circulation. Clinically, they may be classified as background changes, maculopathy and proliferative disease. Any may be present at any level of retinopathy in the same patient, but not all cause visual loss.
Sight-threatening diabetic retinopathy is one of the most common complications of diabetes and is the most common cause of vision loss in the under 65 years age group in developed countries, as a result of non-resolving vitreous hemorrhage, traction retinal detachment or diabetic maculopathy. Sight-threatening diabetic retinopathy refers to diabetic complications affecting the retina that predictably lead to severe loss of vision. These changes include non-resolving vitreous hemorrhage, tractional retinal detachment, or retinal edema.
The following is a description of characteristic anatomical and physiological changes which occur in the retinal circulation in diabetes.
Microaneurysms and intra-retinal microvascular abnormalities (IRMA) are found adjacent to areas of retinal capillary non-perfusion. Infarcts of the nerve fiber layer (sometimes called cotton-wool spots) and intra-retinal hemorrhages are also included in BDR, as are any areas where fluid swells the retina (retinal edema) which does NOT threaten the central vision. Background retinopathy is not associated with visual loss and does not therefore require treatment.
The macula lies at the posterior pole of the retina and is specialized for color and accurate vision (high visual acuity) at the fovea which lies centrally in the macula. It may be altered structurally by fluid or lipids collecting in the retina from localized for widespread leaking retinal capillaries (focal or diffuse maculopathy respectively) or by non-perfusion of para-foveal capillaries (ischemic maculopathy). Additionally, retinal traction, macular hemorrhage or macular hole formation will reduce visual acuity.
As areas of capillary non-perfusion enlarge new vessels arise from the venous circulation at the optic disc (NVD: new vessels at the disc) or from branch retinal veins (NVE: new vessels elsewhere).
Prior to the formation of frank new vessels, there is a pre-proliferative stage which is recognized clinically, as indicated by dilated, beaded retinal veins and signs of widespread retinal non-perfusion. Once the pre-proliferative stage has been identified, the risk of developing PDR in one year is 52% and of developing xe2x80x98high-risk, PDR in five years is 60% (ETDRS12, 1991).
High-risk proliferative diabetic retinopathy is a characteristic appearance of the retina in which 50% of the patients will develop severe visual loss within 5 years. Severe visual loss is a fall of 3 or more lines of visual acuity on the eye chart on two separate occasions 3 months apart. The characteristic retinal findings that would qualify for the diagnosis of high-risk PDR include one or more of the following:
1. New vessels at the disc (NVD) that involve at least xc2xc to ⅓ of the disc area. This is diagnosed by comparison to Standard Photograph 10A of the Modified Airlee House Classification of Diabetic Retinopathy [DRS Report No. 7, 1981];
2. NVD less than xc2xc of the disc area if fresh vitreous hemorrhage is present; and
3. New vessels elsewhere (NVE) greater than or equal to xc2xd the disc area in size if fresh vitreous hemorrhage is present.
Appropriately timed laser treatment can reduce the two year incidence of severe visual loss (SVL) in xe2x80x98high-risk, proliferative disease by 50% and the incidence of moderate visual loss (MVL) over two years in those with macular edema by 50%. [DRS8, 1981; ETDRS1, 1985; ETDRS9, 1991].
Initially, these new vessels leak and are fragile, with the resulting vitreous hemorrhage causing a sudden, often profound, loss of vision. The blood may resolve over the following weeks, but it may become organized into a dense opaque mass in the vitreous which does not resolve.
Fibrous tissue appears adjacent to these new vessels, which subsequently contracts, leading to tractional retinal detachments. New vessels may also grow across the iris and prevent fluid draining from the eye, causing rubeotic glaucoma.
Approximately 16 million people in the United States have diabetes mellitus, of which 50% are diagnosed. Of these, 10-15% have insulin-dependent diabetes mellitus (Type I; IDDM) which is usually diagnosed before the age of 40 years of age. The majority have non-insulin dependent diabetes (Type II DM; NIDDM), some of which may be treated with insulin. From the Wisconsin epidemiological study of diabetic retinopathy, duration of diabetes is associated with increasing incidence of diabetic retinopathy [Klein et al, 1994]. Other risk factors include age less than 30 at diagnosis, being male, raised glycosylated hemoglobin (HbA1c), hypertension, hyperlipidemia, pregnancy and diabetic nephropathy.
Genetic testing is now possible (see U.S. Pat. Nos. 4,582,788 and 5,110,920) for diseases associated with, or caused by, one to two genes, once the genes are identified, to determine the risk of a person carrying a given gene for the disease (see for example U.S. Pat. Nos. 4,623,619, 4,801,531, 4,666,828 and 5,268,267).
U.S. Pat. Nos. 5,039,606 and 5,196,308, as well as Pociot et al. (1992) disclose methods of identifying people who are at risk for developing diabetes. However, these references do not provide methods of determining among diabetics those who are at risk for developing sight-threatening retinopathy. Ko et at. (1995) do provide a single factor, a polymorphic marker at the 5xe2x80x2 end of the aldose reductase gene, that is associated with early onset of diabetic retinopathy in Chinese patients with NIDDM. However, this provides only one marker, which as shown in the Ko et al. reference does not identify all persons at risk. Additionally, the population studied is not the same ethnic population as found in the United States and much of Europe, therefore the data may not be completely applicable to US and European populations.
The pathogenesis of retinal complications from diabetes is not well understood. Michelson (1940) however, proposed that as diabetes produces destruction of blood vessels in the retina, localized areas of ischemia develop resulting in the release vasoproliferative factors. Research in recent years has focused on growth factors that stimulate vascular proliferation, especially basic fibroblast growth factor (BFGF) and vascular endothelial growth factor (VEGF) which are modulated by hypoxia. [Adamis et al., 1994; Aiello et al., 1994; Koyama et al., 1994; Lowe et al., 1995; Malecaze et al., 1994; Mansfield et al., 1994; Murata et al., 1995; Peler etal., 1995; Pfeiffer, 1995; Pfeiffer and Schatz, 1995].
Genetic testing (also called genetic screening or genotyping) can be defined broadly as the testing of nucleic acid of a patient in an analytical capacity to determine if a patient contains mutations (or alleles or polymorphism) that either cause or increase susceptibility to a disease state or are in xe2x80x9clinkage disequilibriumxe2x80x9d with the gene causing a disease state.
Linkage disequilibrium refers to the tendency of specific alleles to occur together more frequently than would be expected by chance. Alleles at given loci are in equilibrium if the frequency of any particular set of alleles (or haplotype) is the product of their individual population frequencies. The cause of disequilibrium is often unclear. It can be due to selection for certain allele combinations, or to a recent admixture of genetically heterogeneous populations. In addition, in the case of markers that very tightly link to a disease gene, an association of an allele (or a group of linked alleles) with the disease gene is expected if the disease mutation occurred in the recent past, so that sufficient time has not elapsed for equilibrium to be achieved through recombination events in that small chromosomal region.
The early detection of a predisposition to genetic diseases presents the best opportunity for medical intervention. Early genetic identification of risk may improve the prognosis for a patient through supervision and early intervention before the clinically detectable disorder occurs.
In cases where patients with similar symptoms are treated with variable success, sophisticated genetic testing can differentiate individual patients with subtle or undetectable differences and can lead to more suitable individual treatments. Early intervention may involve methods such as gene therapy or treatment with IL-1 modulators. With the development of genetic testing, it is now possible to identify gene mutations that indicate a propensity to develop disease, even when the disease is of polygenic origin. The number of diseases that can be identified by molecular biological methods continues to grow with increased understanding of the genetic basis of multifactorial disorders (see, e.g., U.S. Pat. Nos. 4,582,788; 5,110,920; 4,801,531; 4,666,828; and 5,268,267).
The IL-1 gene cluster is located on the long arm of human chromosome 2 (2q13) and contains at least the genes for IL-1xcex1 (IL1A), IL-1xcex2 (IL1B), and the IL-1 receptor antagonist (IL1RN) within a region of 430 Kb (Nicklin, et al., Genomics 19: 382-4 (1994)). The agonist molecules, IL-1xcex1 and IL-1xcex2, have potent pro-inflammatory activity and are at the head of many inflammatory cascades. Their actions, often via the induction of other cytokines such as IL-6 and IL-8, lead to activation and recruitment of leukocytes into damaged tissue, local production of vasoactive agents, fever response in the brain and the hepatic acute phase response. All three IL-1 molecules bind to type I and to type II IL-1 receptors, but only the type I receptor transduces a signal to the interior of the cell. In contrast, the type II receptor is shed from the cell membrane and acts as a decoy receptor. The receptor antagonist and the type II receptor, therefore, are both anti-inflammatory in their actions.
Certain alleles from the IL-1 gene cluster are known to be associated with particular disease states. For example, IL1RN allele 2 is associated with coronary artery disease, osteoporosis, nephropathy in diabetes mellitus (Blakemore, et al., Hum. Genet. 97(3): 369-74 (1996)), alopecia areata (Cork et al., J. Invest. Dennatol. 104(5 Supp.): 15S-16S (1995)), Graves disease (Blakemore, et al., J. Clin. Endocrinol. 80(1): 111-5 (1995)), systemic lupus erythematosus (Blakemore, et al., Arthritis Rheum. 37: 1380-85 (1994)), lichen sclerosus (Clay, et al., Hum. Genet. 94: 407-10 (1994)), and ulcerative colitis (Mansfield, et al., Gastoenterol 106(3): 637-42 (1994)). The IL1B(TaqI) allele 2 from marker+3953 of IL1B is also associated with psoriasis and insulin dependent diabetes in DR3/4 patients (di Giovine, et al., Cytokine 7: 606 (1995); Pociot, et al., Eur J. Clin. Invest. 22: 396-402 (1992)), and severe periodontal disease (kornman, et al. J. Chin. Periodon. 24:72 (1997)).
IL-1 has been shown to play a role in the pathogenesis of Type I diabetes (Dinarello, 1991; Todd, 1990, Mandrup-Poulsen et al., 1993). There is no reported evidence to support a role for IL-1 in either proliferative diabetic retinopathy or maculopathy.
There has been a report on the purported role of cytokines in angiogenesis (Hu and Fan, 1995; Hu et al., 1994). However, Franks et al. [1992]) investigated IL-1, IL-6, IL-2 and TNF in vitreous and aqueous aspirates from eyes undergoing vitrectomy for the treatment of various eye diseases. IL-6 was observed in 6 samples from eyes with diabetic retinopathy (range=5 to 480 pg/ml). Although IL-1 was detected in 80% of all the samples investigated, concentrations of this cytokine greater than 3pg/ml were only observed in one specimens from eyes with uveitis (5 and 20 pg/ml, respectively) and two samples from eyes with diabetic retinopathy (3 and 31 pg/ml, respectively). TNF was present in 1 sample from eyes with diabetic retinopathy (4 pg/ml), and IL-2 was detected in 2 samples from eyes with diabetic retinopathy. The authors concluded that the cytokine presence indicated that a local inflammatory processes may be switched on during many eye diseases and may amplify the clinical signs. The authors did not suggest and their data does not show any primary role for cytokines in the clinical severity of retinopathy. These authors have not reported any follow-up to this observation, and literature searches revealed no other studies that examined cytokines in diabetic retinopathy. The focus in the literature has therefore remained that the dominant factor in controlling clinical outcome is mediated by glucose metabolic factors.
Genetic variation at the multiple loci controlling immune responses, particularly cytokines, has been a factor in determining susceptibility to diseases with inflammatory components. However, diabetic retinopathy is not an inflammatory condition. As discussed above given the tissue changes seen in retinopathy some local production of cytokines can be expected as was seen by Franks et al. [1992], but this is not an inflammatory condition.
It was therefore unexpected to find for a condition that is not primarily inflammatory in nature, both clinically-significant macular edema (maculopathy; CSME) and proliferative diabetic retinopathy (PDR), associated with cytokine gene polymorphisms. The use of combinations of different cytokine polymorphisms indicates those patients who require closer observation and a more aggressive approach to retinopathy when it occurs.
Therefore, it was an objective of the present invention to determine genetic predictors for the risk of developing sight-threatening diabetic retinopathy. It would be useful to identify the genotypes and thereby identify diabetic patients who are susceptible to sight-threatening retinopathy so that early and aggressive monitoring and therapy can be initiated. Further, those patients who may be at higher risk by having the risk factor set forth in the Ko et al. paper and an additional predisposition based on the present invention need to be identified.
According to the present invention, a method for predicting risk of sight-threatening diabetic retinopathy is disclosed. The method includes the steps of isolating DNA from a patient and determining the DNA polymorphism pattern of the genes that code for IL-1xcex1, IL-1xcex2 and IL-1ra. The identified pattern from patients may be compared with controls of known DNA polymorphism patterns thereby identifying patients carrying a genetic polymorphism pattern associated with increased risk of sight-threatening diabetic retinopathy. Patients so identified can then be treated more aggressively in the early stages of diabetes to reduce or prevent the occurrence of severe retinopathy which leads to loss of sight.
The present invention further discloses a kit for the identification of a patient""s genetic polymorphism pattern associated with increased risk of developing sight-threatening diabetic retinopathy. The kit includes DNA sample collecting means and means for determining a genetic polymorphism pattern. The kit may also comprise control samples or standards so that a patient""s risk of sight-threatening diabetic retinopathy can be determined.